Spirulina and Reactive Nitrogen Species: Peroxynitrite, iNOS, and Nitrosative Stress

NO Synthesis: Three NOS Isoforms

Nitric oxide synthases (NOS1/nNOS, NOS2/iNOS, NOS3/eNOS) convert L-arginine plus O2 to NO plus L-citrulline using NADPH, FAD, FMN, BH4, CaM, and haem. NOS3 (endothelial) and NOS1 (neuronal) are constitutively expressed and Ca2+/CaM-activated, producing low physiological NO for vasodilation and neurotransmission. NOS2 (inducible) is transcriptionally induced by NF-kB and IRF-1 in macrophages, epithelial cells, and hepatocytes in response to LPS, IL-1beta, IFN-gamma, and TNF-alpha, producing high-output NO (>1 micromol/L) for cytotoxic pathogen killing, but also bystander tissue damage.

Peroxynitrite Formation and Targets

When high-output iNOS-derived NO meets O2 radical generated by NADPH oxidase or mitochondria, peroxynitrite (ONOO-) forms at near diffusion-limited rates (k approximately 10^10 per M per s). ONOO- is a potent oxidant that: (1) nitrates tyrosine residues (3-nitrotyrosine) on proteins including prostacyclin synthase (Tyr430), Mn-SOD2 (Tyr34, inactivating it), and alpha-synuclein; (2) oxidises BH4, uncoupling eNOS to produce more O2 radical (vicious cycle); (3) nitrosates/nitrates mitochondrial DNA; (4) activates PARP-1 (via DNA strand breaks), depleting NAD+.

PCB and iNOS Suppression via NF-kB

The NOS2 gene contains NF-kB binding sites essential for transcriptional induction. PCB, via Keap1 alkylation and Nrf2 activation, promotes HO-1 expression, and HO-1- derived CO inhibits NF-kB nuclear translocation. PCB also directly inhibits IKKbeta activity in macrophage models (IC50 approximately 10-20 micromol/L), reducing IkBa phosphorylation and NF-kB-driven iNOS transcription. The result is reduced iNOS protein and lower peak NO flux, shifting the system away from ONOO--generating conditions.

SOD2 Protection and the O2 Radical / NO Ratio

The rate of ONOO- formation depends on [NO] times [O2 radical]. Increasing SOD2 activity to reduce O2 radical concentration competes with ONOO- formation. However, nitration of SOD2 Tyr34 by ONOO- inactivates the enzyme, creating a self-amplifying loop. Spirulina's Nrf2-driven Mn-SOD induction (SIRT3-deacetylation also activating SOD2) and AMPK-mitochondrial biogenesis increasing SOD2 protein mass collectively raise the threshold of O2 radical concentration needed for significant ONOO- generation.

eNOS Uncoupling and BH4

When BH4 is oxidised to BH2 (by ONOO- or DHPR deficiency), eNOS becomes uncoupled: the reductase domain transfers electrons to O2 rather than to L-arginine, generating O2 radical instead of NO, which exacerbates both ROS and ONOO- production. Restoring the BH4:BH2 ratio via DHPR (dihydrobiopterin reductase) or by providing antioxidant capacity to prevent BH4 oxidation is therapeutic. Spirulina's Nrf2-driven NADPH generation (via G6PD, ME1) provides reductive equivalents for DHPR, partially maintaining BH4 availability and eNOS coupling.

PARP-1 and NAD+ Depletion

ONOO--induced DNA strand breaks activate poly(ADP-ribose) polymerase 1 (PARP-1), which consumes NAD+ to synthesise PAR chains as a DNA damage signal. Massive PARP activation (parthanatos) depletes cellular NAD+, collapsing glycolysis and OXPHOS, a necrotic cell death mechanism in ischaemia-reperfusion and severe inflammation. Spirulina's dual action, NF-kB suppression reducing iNOS and therefore ONOO-, and AMPK/NAMPT-driven NAD+ repletion, may attenuate parthanatos in ischaemic tissue. This mechanism is relevant to the cytoprotective effects observed in animal stroke models.